Cattle beta-casein gene targeting vector using homologous recombination

ABSTRACT

The present invention relates to a bovine beta-casein gene targeting vector comprising (1) a first region having a length of 5 to 12 kb which is homologous to the promoter and its flanking nucleic acid sequences of bovine beta-casein gene, and comprising exon 1, intron 1, and exon 2 of bovine beta-casein gene; (2) a region for cloning a nucleic acid coding for desired proteins; (3) a region for coding a positive selection marker; (4) a second region having a length of 2.8 to 3.5 kb which is homologous to the nucleic acid sequences of bovine beta-casein gene, and comprising exon 5, 6, 7 and 8, and intron 5, 6 and 7 of bovine beta-casein gene; wherein the nucleic acid segment corresponding to the first region is located upstream to the nucleic acid segment corresponding to the second region in the 5′-3′ arrangement of beta-casein gene. The present invention also relates to method producing the transgenic cattle which is bovine beta-casein gene-targeted with a gene coding a desired protein using the said vector and obtaining a large scale of a desired protein from the milk of the said transgenic cattle.

TECHNICAL FIELD

The present invention relates to bovine beta-casein gene targetingvectors that function through homologous recombination, bovine somaticcells that are gene-targeted with the said vectors and embryos that arenuclear-transferred with the said bovine somatic cells. The presentinvention also relates to methods for producing amounts of desiredproteins from the milk of the transgenic cattle that are prepared byimplanting the said embryos.

BACKGROUND ART

Our continuous efforts in animal genomics to identify genes and theirfunctions have made it possible to generate transgenic animals.Transgenic animals have been created great commercial value inindustries such as biomedicine and agriculture. Various techniques suchas microinjection, viral transfection, sperm vector, application ofembryonic stem (ES) cells and somatic cell nuclear transfer (SNCT) havealso been developed to prepare transgenic animals or so calledgenetically-modified animals.

The microinjection method, which injects a DNA molecule into the male orfemale pronucleus of fertilized eggs (Harbers et al., Nature.,293(5833); 540-2, 1981; Brinster et al., Cell., 27; 223-231, 1981;Gordon et al., Proc Natl Acad Sci USA., 77(12); 7380-7384; Costantini etal., Nature., 294(5836); 92-94), has been widely employed to producetransgenic animals (Hammer et al., Nature., 315(6021); 680-683, 1985;van Berkel et al., Nat. Biotechnol., 20(5); 484-487, 2002; Damak et al.,Biotechnology (NY), 14(2); 185-186, 1996). However, the efficiency ofthis technique in the production of transgenic animals is very low inthat only 2-3% of the injected eggs give rise to transgenic offspring(Clark et al., Transgenic Res., 9; 263-275, 2000). The production oftransgenic animals using the microinjection method is also alabor-intensive and costly procedure requiring large numbers of animalsand facilities (Brink et al., Theriogenology, 53; 139-148, 2000).Another disadvantage of this technique is that it cannot control theintegration sites and the copy numbers of the inserted genes. Theresulting random integration sometimes leads to a low or nonspecificexpression of the transgenes. It was further reported that theunregulated expression of certain transgenes sometimes cause lethalityin the embryonic development (Wei et al., Annu Rev Pharmacol Toxicol.,37; 119-141, 1997).

The retrovirus-mediated method is also widely used in order togenetically manipulate animals (Soriano et al., Genes Dev., 1(4);366-375, 1987; Hirata et al., Cloning Stem Cells., 6(1); 31-36, 2004).Under this technique, the desired gene sequences are introduced into theanimal genomes by using virus-mediated vectors. Although viraltransformation is more efficient than pronuclear injection, randominsertion of the foreign genes and mosaicism are entailed due tomultiple integrations (Piedrahita et al., Theriogenology, 53(1);105-116, 2000). Additionally, the maximum size of the introduced genesis usually limited to approximately 7 kb and there is concerning of thepotential interference caused by virally encoded proteins (Wei et al.,Annu Rev Pharmacol Toxicol., 37; 119-141, 1997; Yanez et al., GeneTher., 5(2); 149-159, 1998).

To circumvent the problems referred to above, a gene targeting techniquethat can insert or remove a DNA segment at a specific location wasdeveloped. The gene targeting technique was first applied to the mouseembryonic stem cells to study gene function. Mouse embryonic stem cellsare now being used to introduce predetermined genetic modifications intoembryos. A number of specific gene-targeted mice have been producedthrough the manipulation of mouse embryonic stem cells using thetechnique(Brandon et al., Curr Biol., 5(6); 625-634, 1995; Capecchi etal., Science, 244(4910); 1288-1292, 1989; Thompson et al., Cell, 56(2);313-321, 1989; Hamanaka et al., Hum Mol. Genet., 9(3); 353-361, 2000;Thomas et al, Cell, 51(3); 503-512, 1987; te Riele et al., Proc. Natl.Acad Sci USA, 89(11); 5182-5132, 1992; Mansour et al., Nature,336(6197), 348-352, 1988; Luo et al., Oncogene, 20(3); 320-328, 2001).The extension of this gene targeting method to other mammalian species,particularly livestock, could bring numerous biomedical benefits such asmass production of pharmaceutical proteins and animal disease models.

Until now, most recombinant therapeutic proteins have been produced bycell culture systems, which use cells such as yeast, bacteria or animalcells. However, it is difficult to produce proteins in large scale usingcell culture systems because of the limited capacity and high cost.Furthermore, for some proteins, additional steps are required tointroduce proper posttranslational modifications such as glycosylation,γ-carboxylation, hydroxylation and so on (Houdebine et al., TransgenicRes., 9(4-5); 305-320, 2000; Lubo et al., Transgenic Res., 9(4-5);301-304, 2000).

Animal bioreactors that produce valuable or therapeutic proteins havebeen evaluated as efficient and cost-effective expression systems. Inparticular, the large-scale production of therapeutic recombinantproteins from transgenic animals is much more cost-effective compared tothe cell culture system (van Berkel et al., Nat. Biotechnol., 20(5);484-487, 2002). Recombinant proteins produced in animal milk were knownto be post-translationally modified in a way very similar to the humancounterpart proteins (Edmunds et al., Blood, 91(12); 4561-4571, 1998;Velander et al., Proc Natl Acad Sci USA., 89(24); 12003-12007, 1992; vanBerkel et al, Nat. Biotechnol., 20(5); 484-487, 2002).

Cow's milk is composed of approximately 88% water, 3.3% protein and theremaining carbohydrates and fat. The caseins, comprising 80% milkprotein, are divided into four groups, alpha S1, alpha S2, beta andkappa casein. Beta casein is the most abundant protein in milk and isexpressed in a concentration of 10 mg/ml in bovine milk (Brophy et al.,Nat. Biotechnology., 21(2); 157-162, 2003).

The somatic cell nuclear transfer (SCNT) technique is more efficient wayto make transgenic animals compared to the microinjection method becausealmost all of the cloned animals derived from transformed somatic cellsare transgenic. animals (Brink et al., Theriogenology, 53; 139-148,2000). It is also possible to predetermine the sex of animals and createa genetically homogeneous herd in order to produce a uniform product(Lubo et al., Transgenic Res., 9(4-5); 301-304, 2000; van Berkel et al.,Nat. Biotechnol., 20(5); 484-487, 2002).

Until now, ES cells and vector constructs for targeting a specific genehave been considered as prerequisite elements to generate gene-targetedanimals. However, there is a limitation in the use of ES cells fromlarge livestocks, although some studies have developed ES-like cells inpig and cattle (Doetschman et al., Dev Biol., 127(1); 224-227, 1988;Stice et al., Biol Reprod., 54(1); 100-110, 1996; Sukoyan et al., MolReprod Dev., 36(2); 148-158, 1993; Iannaccone et al., Dev Biol., 163(1);288-292, 1994; Pain et al., Development, 122(8); 2339-2348, 1996;Thomson et al., Proc Natl Acad Sci USA., 92(17); 7844-7848, 1995;Wheeler et al., Reprod Fertil Dev., 6(5); 563-568, 1994).

Instead, the use of normal somatic cells as nuclear donor cells has beensuggested as an efficient and practical method to produce transgeniccattle (Brophy et al., Nat. Biotechnol., 21(2); 157-162, 2003; Cibelliet al., Science, 280(5367); 1256-1258, 1998; Campbell et al., Nature,380(6569); 64-66, 1996; Wilmut et al., Experientia, 47(9); 905-912,1997; Denning et al., Cloning stem cells, 3(4); 221-231, 2001),suggesting the possibility that somatic cells instead of embryonic stemcells can be used for targeting specific genes.

With the application of SCNT technique, promoter regions of milk proteingenes have been used to direct the expression of recombinant protein inthe milk of transgenic large animals (Schnieke et al. Science,278(5346); 2130-2133, 1997; Baguisi et al., Nat. Biotechnol., 17(5);456-461, 1999; Brophy et al., Nat. Biotechnol., 21(2); 157-162, 2003).However, the use of this technique in farm animals is still notpractical unless the problems of low expression and/or ectopicexpression due to the random insertion of genes are solved. The ectopicexpression of foreign protein causes early embryonic lethality and isparticularly severe in nervous system, as most nervous system structuresdevelop in late embryonic and early postnatal stages (Gao et al.,Neurochem Res., 24(9); 1181-1188, 1999). To eliminate or reduce theseside-effects, a new method that allows the foreign protein to beexpressed only during the lactation period and strictly in the mammarygland have been invented (Houdebine et al., Transgenic Res., 9(4-5);305-320, 2000). Gene-targeting, known as the introduction ofsite-specific modification into a genome by homologous recombinationevent, is a powerful tool for tissue-specific expression of recombinantproteins (Muller et al., Mech Dev., 82(1-2); 3-21, 1999; Clark et al., JMammary Gland Biol Neoplasia., 3(3); 337-350, 1998).

The generation of a first knock-in ovine has opened the door to make itpossible to produce therapeutic foreign protein-targeted large animals(McCreath et al., Nature, 405(6790); 1066-1069, 2000). The COLT-2targeting vector which has homology regions for COL1A1 gene that ishighly expressed in fibroblasts was developed, allowing thepromoter-trap enrichment of gene-targeting events. AATC2 transgenecomprised of human al-antitrypsin (AAT) complementary DNA within anovine beta-lactoglobulin (BLG) expression vector was designed to directexpression in the mammary gland, having separate transcription unit. Theamount of AAT secreted from targeted lambs was 37-fold more than thatfrom the sheep with multiple and random integration of the genes(McCreath et al., Nature, 405(6790); 1066-1069, 2000). Thus, genetargeting is now regarded to be the most powerful method to producelarge amount of therapeutic protein. However, the application of thepromoter-trap targeting vector is limited to transcriptionally activegenes in the somatic cells. In general, transcriptionally active genesare more amendable to gene targeting than silent genes because they havea higher frequency of homologous recombination (Kuroiwa et al., Nat.Genet., 36(7); 775-780, 2004).

Milk proteins are expressed in a tissue-specific manner in the mammarygland. An over-expression of foreign protein is possible without causinglethality in embryonic or post-natal development by manipulating a genecoding of the milk proteins. Among such proteins, beta-casein would beone of the best candidates since it is expressed abundantly. The bovinebeta-casein exists as a single copy gene in total genome and is notknown to be expressed in the somatic cells except for those in themammary gland. The targeting of foreign genes into the beta-casein gene,which is not expressed in the normal somatic cells, cannot be carriedout by vectors utilizing the promoter trap.

Based on this background, the present inventors have created targetingvector cassettes specific for the bovine beta-casein gene, vectorsinserted with a foreign gene using the said cassettes, bovine somaticcell introduced with the said vector, and nuclear-transferred embryowith the said bovine cell. The present inventors found that the foreigngene was correctly targeted to the beta-casein gene of bovine genomicDNA and confirmed the targeting events with the said vector were highlyefficient. Therefore, the present inventors have been completedtransgenic cattle which could produce a large scale of desiredtherapeutic protein using the said bovine beta-casein gene targetingvector cassette.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a bovine beta-caseingene targeting vector comprising (1) a first region having a length of 5to 12 kb which is homologous to the promoter and its flanking nucleicacid sequences of bovine beta-casein gene, and comprising exon 1, intron1, and exon 2 of bovine beta-casein gene; (2) a region for cloning anucleic acid coding for desired proteins; (3) a region for coding apositive selection marker; (4) a second region having a length of 2.8 to3.5 kb which is homologous to the nucleic acid sequences of bovinebeta-casein gene, and comprising exon 5, 6, 7 and 8, and intron 5, 6 and7 of bovine beta-casein gene; wherein the nucleic acid segmentcorresponding to the first region is located upstream to the nucleicacid segment corresponding to the second region in the 5′-3′ arrangementof beta-casein gene.

It is another object of the present invention to provide a bovinesomatic cell which is bovine beta-casein gene-targeted with the saidtargeting vector.

It is another object of the present invention to provide an embryo whichis nuclear-transferred with the said somatic cell.

It is another object of the present invention to provide a method forpreparing bovine beta-casein gene-targeted somatic cell, which comprisesthe steps of (1) introducing the above vector into a bovine somaticcell; (2) occurring homologous recombination events in the bovinesomatic cell; and (3) selecting the bovine beta-casein gene-targetedsomatic cell.

It is another object of the present invention to provide a method forpreparing transgenic cattle, which comprises the steps of (1)introducing the above vector into a bovine somatic cell; (2) occurringhomologous recombination events in the bovine somatic cell; (3)selecting the bovine beta-casein gene-targeted somatic cell; (4)introducing the above gene-targeted cell into a nuclear-removed bovineoocyte to produce nuclear-transferred embryo; and (5) implanting theabove embryo into a surrogate to produce cloned transgenic cattle.

It is yet another object of the present invention to provide a methodfor preparing a large scale of desired therapeutic proteins from milk ofcloned cattle produced using the above method.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts 18.8 kb pBCKI I vector cassette targeting the bovinebeta-casein gene, derived from a pBluescript II SK(+) plasmid. Thevector also includes Sac II, Not I, and BamH I restriction sites infront of neo gene and BamH I site at the back of short arm region.

FIG. 2 depicts 14.8 kb pBCKI II vector cassette targeting the bovinebeta-casein gene, derived from a pGEM-7Zf(+) plasmid (Promega).

FIG. 3 depicts 160.1 kb pBCKIDT II vector cassette targeting the bovinebeta-casein gene, derived from pGEM-7Zf(+) plasmid. The vector alsoincludes Sac II, NotI, and BamH I restriction sites in front of neo geneand BamH I site at the back of short arm region. In addition, 1.3 kbdiphtheria toxin (DT) gene as a negative selection marker is inserted.

FIG. 4 shows homologous recombination between pBCKI I vector andbeta-casein gene in genome. Double crossover results in the replacementof the endogenous beta-casein gene with the sequences in the vectorcassette.

FIG. 5 shows homologous recombination between pBCKI II vector and abeta-casein gene in genome. A partial sequence of a beta casein genelocus in genome is replaced with a partial sequence in a vector cassetteas result of a double crossover event.

FIG. 6 shows homologous recombination between pBCKIDT II vector and abeta-casein gene in genome. Double crossover results in the replacementof the endogenous beta-casein gene with the sequences in the vectorcassette. As results of homologous recombination events, DT gene isdeleted.

FIG. 7 shows PCR screening strategy used to identify gene targetingevents at the bovine beta-casein locus and the DNA sequences spanningthe junctions of the targeted beta-casein locus and the restrictionsites in the targeting vector cassettes, pBCKI I and pBCKI II. The upperportion shows the approximate position of two primers used to amplifythe 4 kb fragment, from the neo gene region unique to the beta-caseingene targeting vectors to the endogenous beta-casein locus which is notused in the vector cassettes. Sequence A shows a portion of DNA sequencespanning the junction between the long arm region and the neo gene.Sequence B shows a portion of DNA sequence spanning the junction betweenthe neo gene and the short arm region. The position and orientation ofthe 5′ PCR primer is shown. Sequence C shows a portion of DNA sequencespanning the junction between the short arm region and the endogenousbeta-casein gene locus which is excluded from the BCKI I and BCKI IIvectors. The position and orientation of the 3′ PCR primer is shown.

FIG. 8 shows PCR screening strategy used to identify gene targetingevents at the bovine beta-casein locus and the DNA sequences spanningthe junctions of the targeted beta-casein locus and the restrictionsites in the targeting vector cassette, pBCKIDT II. The upper portionshows the approximate position of two primer sets used to amplify the 4and 3.4 kb fragments, from the neo gene region unique to the beta-caseingene targeting vectors to the region endogenous beta-casein locus whichis not used in the vector cassettes. Here, after amplifying 4 kb PCRfragments using the first primer set, 3.4 kb PCR fragments wereamplified using the second primer set for a small volume of DNA samples.Sequence A shows a portion of DNA sequence spanning the junction betweenthe long arm region and the neo gene. Sequence B shows a portion of DNAsequence spanning the junction between the neo gene and the short armregion. The positions and orientations of the 5′ PCR primers are shown.Sequence C shows a portion of DNA sequence spanning the junction betweenthe short arm region and the endogenous beta-casein gene locus which isexcluded from the BCKI I and BCKI II vectors. The positions andorientations of the 3′ PCR primers are shown.

FIG. 9 shows the pBC10 vector constructed in our previous study (Kim eta 1., J Biochem (Tokyo)., 126(2); 320-5, 1999). The pBC10 vector isbased on a pBluescript II SK vector backbone and includes the bovinebeta-casein promoter region, which is 10 kb in length. The promoterregion contains 8 kb of the 5′-flanking sequence of gene, theuntranslated exon 1 and 2 (vertical open boxes), and 2 kb of intron 1 ofbovine beta-casein gene. The promoter has restriction enzyme sites forSac I, Aat II, and Sac II, but there is no recognition sequences ofother restriction enzymes such as Sma I, BamH I, Sal I, Spe I and Cla I.

FIG. 10 shows the isolated 10 kb DNA fragment from the pBC10 vectordigested with Sac I and Sac II restriction enzymes. The isolated 10 kbDNA fragment is used for a long arm of the pBCKI I vector cassette.

FIG. 11 shows the isolated 6 kb DNA fragment from the pBC10 vectordigested with Aat II and Sac II restriction enzymes. The isolated 6 kbDNA fragment is used for a long arm of the pBCKI II vector cassette.

FIG. 12 shows construction procedures of the pBC3.1 vector applied forthe short arm of pBCKI I and pBCKI II vector cassettes. The 3.2 kb DNAfragment including exon 5, 6, 7 and 8 of bovine beta-casein gene wasprepared by PCR amplification using the primer set which has Xho I andSal I sites (bold characters) from the bovine chromosomal DNA. Theamplified PCR fragment was digested with Xho I and Sal I restrictionenzymes and then ligated into the Sal I restriction enzyme site ofpGEM-T vector (Promega). The 3.2 kb DNA fragment in the PGEM-T vectorwas digested with Hinc II and Sal I restriction enzymes and then ligatedinto Sal I site of pBluescript II SK(+) vector.

FIG. 13 shows the procedure obtaining the neo gene including the SV40ori, the early promoter, the neomycin resistance gene, the SV40 earlysplicing region and polyadenylation site from a pMAMneo vector(CLONTECH). The 2.7 kb neo gene fragment digested with BamH Irestriction enzyme was ligated into BamH I site of pBluescript II SK(+)vector. The 2 kb DNA fragment of the neo gene in a pBluescript II SK(+)vector was digested with Bgl II and BamH I restrction enzymes andligated into Bgl II site of pSP73 vector (Promega). The 2 kb fragment ofa pSP73 vector digested with Bgl II and EcoR V restriction enzymes wasthen re-ligated into Bgl II and EcoR V sites of the pBluescript II SK(+)vector containing the 0.7 kb neo gene to generate the pneo2.7 vectorconstruct.

FIG. 14 shows the construction procedures of the pBCKI I and pBCKI IIvector cassettes. The DNA fragments corresponding to a positiveselection marker and short arm in pBluescript II SK(+) were digestedwith respective restriction enzymes, and then ligated into the BamH IEcoR V, and Sal I site of pBluescript II SK(+) vector harboring pBC10DNA fragment. The resultant pBCKI I vector includes the 10 kb long arm,the selection marker gene and the short arm. To shorten long arm lengthof the pBCKI I, the DNA fragment digested with Aat II and Sal Irestriction enzymes was ligated into Aat II and Xho I sites ofpGEM7Zf(+) vector. The resultant pBCKI II vector includes the 6 kb longarm, the neo gene, and the short arm.

FIG. 15 shows the procedure obtaining the DT gene, inserted into pBCKIDTII vector, from pKO SelectDT (Lexicon Genetics). Using restrictionenzyme, Rsr II, diphtheria toxin A (DT) gene containing SV40 Poly A andRNA polymerase II promoter was isolated. After klenow-filling theisolated DT gene, it was inserted into Hind II sites of pBluescript IISK(+) vector (Stratagene), generating pSK DT vector. The DT gene in thepSKDT vector was digested with Xho I and EcoR V restriction enzymes andligated into Xho I and Pvu II sites of a pSP73 vector (Promega) togenerate the pSP73DT vector construct.

FIG. 16 shows the construction procedures of the pBCKIDT II vectorcassette. DT gene in pSP73DT vector was digested with restrictionenzyme, Xho I and Sal I and then ligated into Sal I site of pBCKI Ivector cassette, generating pBCKIDT I vector. The resultant pBCKIDT Ivector was digested with Aat II and Sal I and then ligated into Aat IIand Xho I sites of pGEM-7Zf(+) vector (Promega), generating pBCKIDT IIvector cassettes.

FIG. 17 shows introduction of a valuable gene into the pBCKI I vector.As an example of the genes for therapeutic proteins, humanthrombopoietin (hTPO) cDNA was inserted into the invented PBCKI I vectorcassettes. The 1.0 kb of the hTPO cDNA coding for the full length formwas amplified by PCR in our previous study, and the poly(A) additionalsignal sequence of the bovine growth hormone (bGH) gene, which is 300 bpin length, was ligated into the Kpn I site to downstream of the hTPOcDNA fragment (Sohn, DNA Cell Biol., 18(11); 845-852, 1999). The 1.3 kbDNA fragment including hTPO cDNA and bGH gene was inserted into Sac IIand Not I sites of the pBCKI I vectors, generating the 20.1 kb pBCTPOKII vector construct.

FIG. 18 shows introduction of the hTPO cDNA into pBCKI II vectorcassette. The resultant 16.1 kb pBCTPOKI II construct was generated andthe procedures were the same as described in FIG. 17.

FIG. 19 shows introduction of the hTPO cDNA into pBCKIDT II vectorcassette. As a result, 17.4 kb pBCTPOKIDT II construct was generated andthe construction procedures were the same as described in FIG. 17.

FIG. 20 shows the linearized pBCTPOKI I vector to be used fortransfection after Sal I digestion.

FIG. 21 shows the linearized pBCTPOKI II vector to be used fortransfection after Aat II digestion.

FIG. 22 shows the linearized pBCTPOKIDT II vector to be used fortransfection after deleting a plasmid vector by Aat II and Cla Idigestion.

FIG. 23 shows the linearized pBCTPOKI II vector to be used fortransfection after deleting a plasmid vector by Aat II and Cla Idigestion. Whereas the pBCTPOKI II vector, described in FIG. 21, wassimply linearized and transfected into cells with a plasmid vector, thepBCTPOKI II vector, described here, was transfected into cells afterdeleting a plasmid vector by two restriction enzyme digestion.

FIG. 24 is photographs showing morphologies of bovine embryonicfibroblasts (bEF) and bovine ear skin fibroblasts (bESF). A and B arenon-transfected (normal) cells, bEF and bESF, respectively, cultured toconfluence. C and D show the colony formed after transfection of thetargeting vector into bEF and bESF, respectively. After the targetingvector which has antibiotic resistance gene, neo gene, is introducedinto bEF and bESF using the Lipofectamine™ 2000 reagent (Invitrogen)method, the transfected cells were treated with G418 (Gibco, Invitrogencorporation) and then only the survived colonies were subject toanalyses of the inserted foreign genes.

FIG. 25 shows PCR analysis of the survived colonies after pBCKI IItransfection. The positive signals for the 500 bp PCR fragment indicatedby an arrow represent transfected cell clones, were denoted as “+” andthe negative signals were denoted as “−”. The pBCTPOKI II vector of thepresent invention was used as a positive control and the PCR wasamplified with the primers specific for hTPO gene.

FIG. 26 and FIG. 27 depict construction procedures of the pneoBC3.7vector to be used as the control for long-range PCR analysis. The 591 bpDNA fragment harboring intron 8 and exon 9 corresponding to bases 7888to 8479 of bovine beta-casein gene was prepared separately by PCRamplification using a primer set from the bovine chromosomal DNA. The 3′primer has Xho I site depicted as bold characters. The amplified PCRfragment was ligated into pGEM-T vector to generate the pBC591 vectorconstruct. The pBC591 vector was digested with Sal I and Xho Irestriction enzymes, and then the resultant DNA fragment was ligatedinto Sal I site of the pBC3.1 vector. At the same time, the pneo2.7vector digested with BamH I and EcoR V restriction enzymes, and then theresultant DNA fragment was ligated into the pBC3.1 vector digested withBamH I and EcoR V restriction enzymes to generate the pneoBC3.7 vector.The arrows show primer set for 4 kb long-range PCR analysis.

FIG. 28 shows the results of long-range PCR analyses of the bESF cellcolonies transfected with pBCTPOKI II. The positive signals for the 4 kbindicated by arrows represent targeted cell clones, were detected in twocolonies, “81” and “89”, and the other colonies showed negative signals(−). The pneoBC3.7 vector was used as a positive control.

FIG. 29 shows the results of long-range PCR analyses of the bESF cellcolonies transfected with pBCTPOKI II and pBCTPOKIDT II. The positivesignals for the 3.4 kb indicated by arrows represent targeted cellclones. The first PCR analysis was performed from approximately fivecells and then the second PCR analysis was performed from the first PCRproducts, showing resultant 3.4 kb positive signals (FIG. 8). A is theresults of long-range PCR analyses of cell colonies transfected withpBCTPOKIDT II vector and the number 5, 30, 17, 18, 20, 21, and 26 cellcolonies were identified to be gene-targeted. B is the results oflong-range PCR analyses of cell colonies transfected with pBCTPOKI IIand the number 16 cell colony was identified to be gene-targeted. C isthe results of long-range PCR analyses of wild-type bovine genomic DNAused as a negative control (−) and pneoBC3.7 vector used as a positivecontrol (+).

FIG. 30 shows strategy for Southern blot analysis to identify thetargeted nucleic acid sequences. The BCTPOKI II vector and purifiedgenomic DNAs from transfected cell clones were digested with EcoRI. Thesize of DNA fragments from BCTPOKI II vector (A) and genomic DNA (B)digested with the EcoR I restriction enzyme are 9.2 kb and 9.9 kb,respectively. The bar under the hTPO gene represents the locus of theprobe used for Southern analysis. 500 bp fragments of hTPO cDNA used forthe probe were amplified by PCR.

FIG. 31 shows identification of targeted colonies by Southern blotanalysis. Two colonies, 81 and 89, were identified to be targeted at theendogenous bovine beta-casein gene locus with the pBCTPOKI II vector ofthe present invention. The targeted colonies represented the 9.9 kbfragment and the other colonies, 97, 47, 43 and 34, did not show anysignal. The bovine genomic DNA was used at the various concentrations asa negative control and the 9.2 kb fragments derived from the pBCTPOKI IIvector on various concentrations were used as a positive control.

FIG. 32 shows PCR analysis of nuclear-transferred embryos from 81 cellclone. The positive (+) signals, 356 bp in length, implied that thecloned embryos derived from the somatic cells that were targeted withthe pBCTPOKI II vector of the present invention. The pBCTPOKI II vectorwas used as a positive control. And nested PCR was also performed tore-confirm the positive signals.

FIG. 33 is a photograph showing a 89 cell clone derived fetus enclosedwith a fetal membrane collected at 36 days of gestation (A), the fetalmembrane-removed fetus (B) and cells derived from the fetal membrane(C), the result of the long-range PCR of cells derived from the fetalmembrane (D). The positive signals for the 4 kb indicate that cells,genetically identical to the cloned fetus, were targeted with thepBCTPOKI II vector of the present invention. The pneoBC3.7 vector wasused as a positive control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to preparing bovine beta-casein genetargeting vectors used to generate beta-casein gene-targeted cattlewhich produce amounts of desired therapeutic proteins from milk. Whereasgoat and sheep produce milk to 300 and 200 liters, respectively, a year,cattle produce milk up to 6,300 liter a year. Cattle are optimal speciesthat could be used as animal bioreactors, however, there has been noreport about targeting the desired gene to milk protein genes in bovine.The present invention relates to vector constructs which can target to abovine beta-casein gene of bovine milk protein genes and a methodpreparing beta-casein gene targeted cattle which produce amounts oftherapeutic proteins using the vector constructs of the presentinvention.

In an aspect, the present invention relates to a bovine beta-casein genetargeting vector comprising (1) a first region having a length of 5 to12 kb which is homologous to the promoter and its flanking nucleic acidsequences of bovine beta-casein gene, and comprising exon 1, intron 1,and exon 2 of bovine beta-casein gene; (2) a region for cloning anucleic acid coding for desired proteins; (3) a region for coding apositive selection marker; (4) a second region having a length of 2.8 to3.5 kb which is homologous to the nucleic acid sequences of bovinebeta-casein gene, and comprising exon 5, 6, 7 and 8, and intron 5, 6 and7 of bovine beta-casein gene; wherein the nucleic acid segmentcorresponding to the first region is located upstream to the nucleicacid segment corresponding to the second region in the 5′-3′ arrangementof beta-casein gene.

The term “gene targeting vector”, as used herein, means a vector thatcan remove or insert a desired gene to a specific-genomic locus andincludes a nucleic acid sequence homologous to a particular gene forhomologous recombination. A gene targeting vector of the presentinvention is a beta-casein targeting vector which inserts nucleic acidsequences coding desired proteins to beta-casein gene in genome in the5′-3′arrangement. The terms, vectors and vector cassettes are usedherewith interchangeably, and the vectors can be either circular orlinear form. The gene-targeting vectors of the present invention arebovine beta-casein gene-targeting vectors.

The beta-casein targeting vector of the present invention comprises afirst region and a second region that are homologous to the sequences ofthe beta-casein gene that are located before and after the cloning site.A first region corresponds to the long arm and a second regioncorresponds to the short arm.

Among components of the invented bovine beta-casein gene targetingvectors, particularly, “a first region” and “a second region” areimportant parameters to determine the gene targeting efficiency.

“A first region” of herein, is characterized by having a length of 5 to12 kb which has a DNA sequences homologous to the promoter and itsflanking sequences of bovine beta-casein gene, and comprising exon 1-2and intron 1 of bovine beta-casein gene. A bovine beta-casein genepromoter has been known to be a good promoter to induce high expressionof a foreign protein (Kim et al., J Biochem (Tokyo)., 126(2); 320-325,1999) and is a good candidate to express a large scale of desiredforeign proteins. Preferably, 5.5 kb to 10 kb is preferable as a length.

“A second region” of herein, characterized by having a length of 2.8 to3.5 kb which is homologous to a DNA sequence of bovine beta-casein gene,and comprising exon 5-8 and intron 5-7 of bovine beta-casein gene. 3.0kb to 3.2 kb is preferable as a length.

The first region is located upstream to the second region in the5′-3′arrangement of beta-casein gene.

The term “homology”, as used herein, means the degree of similaritybetween nucleic acid sequences of the first region or a second regionand the endogenous beta-casein gene sequences corresponding to thesefirst and second regions. The sequences are homologous to each otherwhen the sequences exhibit at least 90%, preferably at least 95%sequence identity.

The term “(multiple) cloning site”, as used herein, means a nucleic acidsequence comprising at least two distinct nucleotide sequencespecifically recognized and digested by restriction enzymes to permitinsertion of nucleic acid sequence coding desired proteins.

A desired protein, which can inserted to the MCS on a vector of thepresent invention, may includes all proteins having medical orindustrial application, such as hormones, cytokines, enzymes,coagulation factors, carrier proteins, receptors, regulatory proteins,structural proteins, transcription factors, antibodies, antigens, etc.

Specific examples of the desired proteins include, but not limited to,thrombopoietin, human growth hormone, growth hormone releasing hormone,growth hormone releasing peptide, interferons, interferon receptors,colony-stimulating factor, glucagon-like peptides, G-protein-coupledreceptor, interleukins, interleukin receptors, enzymes, interleukinbinding proteins, cytokine binding proteins, macrophage activatingfactor, macrophage peptide, B cell factor, T cell factor, protein A,suppressive factor of allergy, cell necrosis glycoprotein, immunotoxin,lymphotoxin, tumor necrosis factor, tumor inhibitory factor,transforming growth factor, alpha-1 antitrypsin, albumin,alpha-lactalbimin, apolipoprotein-E, erythroprotein, hyper-glycosylatederythroprotein, angiopoietins, hemoglobin, thrombin, thrombin receptoractivating peptide, thrombomodulin, factor VII, factor VIIa, factorVIII, factor IX, factor XIII, plasminogen activator, fibrin bindingprotein, urokinase, streptokinase, hirudin, protein C, C-reactiveprotein, renin inhibitor, collagenase inhibitor, superoxide dismutase,leptin, platelet derived growth hormone, epithelial growth factor,epidermal growth factor, angiostatin, angiotensin, osteogenic growthfactor, osteogenesis stimulating protein, calcitonin, insulin,atriopeptin, cartilage inducing factor, elcatonin, connective tissueactivator protein, tissue factor pathway inhibitor, follicle stimulatinghormone, luteinizing hormone, FSH releasing hormone, nerve growthhormone, parathyroid hormone, relaxin, secretin, somatomedin,insulin-like growth factor, adrenocorticotrophic hormone, glucagon,cholecystokinin, pancreatic polypeptide, gastrin releasing peptide,corticotropin releasing factor, thyroid stimulating hormone, autotaxin,lactoferrin, myostatin, receptor, receptor antagonist, cell surfaceantigen, virus-derived vaccine antigen, monoclonal antibody, polyclonalantibody, and so on.

The term “selection marker”, as used herein, is required for selectingtransformed cell with the vector. The selection marker gene confersresistances to drugs, nutritional requirement and cytotoxic drugs, orinduces selectable phenotype such as fluorescence and a color deposit.There is a positive selection marker and a negative selection marker.“Positive selection marker” makes cell expressing positive selectionmarkers to survive against selective agent, so that be capable ofconferring positive selection characteristic for the cell expressingthat marker. The positive selection marker includes, but not limited to,neomycin, hygromycin, histidinol dehydrogenase, guaninephosphosribosyltransferase, and so on.

The vector of the present invention comprises negative selection markeras additional component. “Negative selection marker” removes cells withrandom-integration, so that be capable of conferring negative selectioncharacteristic for the cell expressing that marker. The negativeselection marker includes, but not limited to, thymidine kinase (tk),hypoxanthine phosphoribosyl transferase (Hprt), cytosine deaminase,diphtheria toxin (DT), and so on. The negative selection marker locatesat the 5′ terminus of a first DNA sequence or the 3′ terminus of thesecond DNA sequence. Among those negative selection markers, tk and DTis used generally. In the present invention, DT gene was utilized as anegative selection marker. Whereas tk requires treatment of gancyclovir,DT does not require any other treatments. It is also reported thattk-carrying cells treated with gancyclovir show potent ‘bystandereffect’ on co-culturing with unmodified cells and gancyclovir suppressthe growth of cells (Yoshiyasu Kaneko et al., Cancer Letters, 96;105-110, 1995). In those aspects, DT is more preferable than tk.

The positive and negative selection markers of the present inventionhave independent promoter and poly A regions. The promoter includes, butnot limited to, simian virus 40 (SV40) promoter, mouse mammary tumorvirus (MMTV) promoter, human immunodeficiency virus-long terminal repeat(HIV-LTR) promoter, moloney virus promoter, cytomegalovirus (CMV)promoter, epstein-barr virus (FBV) promoter, respiratory syncytial virus(RSV) promoter, RNA polymerase II promoter, β-actin promoter, humanhemoglobin promoter, human muscle creatin promoter, and so on.

The nucleic acid coding the desired protein is integrated to abeta-casein gene locus of cellular genomic DNA in host cell byhomologous recombination, and is expressed in the cell instead of theendogenous beta-casein protein.

To improve efficiency of homologous recombination events, the bovinebeta-casein gene targeting vectors of the present invention havecharacteristics as follows.

The efficiency of gene integration of the nucleic acid coding a desiredprotein into a beta-casein gene locus of cellular genomic DNA has beenshown to have relation with the targeting vector system, especially withthe degree of similarity and length of nucleic acid sequences ofhomologous regions (Scheerer et al., Mol Cell Biol., 14(10)6663-6673,1994; Thomas et al., Cell, 51(3); 503-512, 1987; Hasty et al., Mol CellBiol., 11(11); 5586-5591, 1991; Lu et al., Blood, 102(4); 1531-1533,2003). The present invention maximized the efficiency by optimizing theposition and length of homologous region of the first and secondregions. In addition, to minimize positional interference whenhomologous recombination events occurs, the positive selection markersof the invented vectors were inserted into loci corresponding exon 3-4and intron 2-4 regions of bovine beta-casein gene. In addition, in thepresent invention, multiple cloning sites (MCS) were developed in thebehind of “a first region” to insert foreign protein genes for ease. Inaddition, final vector scheme of the present invention comprising genecoding desired gene for gene targeting is different from the one ofprevious (SHEN Wei et al., Chinese Journal of Biotechnology, 20(3);361-365, 2004). 3 of homology regions for homologous recombinationevents existed in vector of the previous report, because gene codingdesired protein were inserted randomly into a first region of the vector(SHEN Wei et al., Chinese Journal of Biotechnology, 20(3); 361-365,2004). Whereas, only two homology regions for recombination eventexisted in vector of the present invention, because gene coding desiredprotein were inserted into MCS of vector. Accordingly, when the vectorcassette of the present invention is used, construction of the vectorfor gene targeting is simple and targeting efficiency of vector is muchhigher than that of previous one (SHEN Wei et al., Chinese Journal ofBiotechnology, 20(3); 361-365, 2004).

Indeed, the present vector system induced homologous recombinationevents even in the somatic cells that are transcriptionally silent,leading to the stable integration of the foreign protein gene into abeta-casein gene locus.

In a preferred embodiment of this invention, pBCKI I and pBCKI IIvectors, and pBCKIDT I and pBCKIDT II vectors harboring a negativeselection marker were prepared as targeting vectors.

pBCKI I vector, about 18.8 kb in length, was derived from pBluescript IISK(+) plasmid. The first region of pBCKI I vector is about 10 kb inlength and it includes 8 kb sequences harboring bovine beta-caseinpromoter, exon 1, intron 1 and exon 2 of the bovine beta-casein gene. Asecond region of pBCKI I vector is about 3.1 kb in length and itincludes exons 5-8, introns 5-7 and a portion of intron 4 and 8 as shownin FIG. 2. The pBCKI I vector includes a neo gene as a selection marker,SV40 early promoter and polyA sequences. The pBCKI I vector includesthree restriction enzymes sites (Sac II, Not I and BamH I), for theinsertion of nucleic acid coding desired proteins as shown in FIG. 1.

pBCK II vector, about 14.8 kb in length, was derived from pGEM7Zf(+)plasmid. The first region of pBCKI II is about 6 kb in length and itincludes bovine beta-casein promoter having a length of about 4 kb andaxon 1, intron 1 and exon 2 of the bovine beta-casein. A second regionof pBCKI II vector is about 3.1 kb in length and includes exons 5-8,introns 4-7 and a portion of intron 4 and 8 as shown in FIG. 2. ThepBCKI II vector includes a neo gene as a selection marker gene, SV40early promoter and polyA. The pBCKI II vector includes three restrictionenzymes sites (Sac II, Not I and BamH I), for the insertion of nucleicacid coding desired proteins as shown in FIG. 2.

The pBCKIDT I and pBCKIDT II vectors were developed as inserting DT geneinto Xho I and Sal I sites of the pBCKI I and pBCKI II vectors.

The targeting vectors according the present invention can be preparedusing general DNA recombination techniques. Site-specific cleavage andligation can be accomplished by using the enzymes known to the art.

In a specific embodiment, human thrombopoietin (TPO) gene was insertedinto BCKI I and BCKI I (FIGS. 17, 18 and 19). The 1 kb human TPO cDNAfragment flanked by 0.3 kb fragment of bovine growth hormone (bGH) genewas constructed (Sohn et al., DNA Cell Biol., 18(11); 845-852, 1999).The bGH gene was inserted to encode s stable messenger RNA.

Human thrombopoietin is one of the major hemopoietic regulatorsparticipating in series of megakaryocytopoiesis, that is, proliferationand differentiation of megakaryocytes inducing production of platelets.As a primary physiological regulator of platelet production, it plays apivotal role in promoting the proliferation and maturation ofmegakaryocytes. Severe neutropenia and thrombocytopenia are seen inpatients undergoing aggressive chemotherapy and bone marrowtransplantation for hematologic malignancies and solid tumors (Lok etal., Stem Cells, 12(6); 586-598, 1994; Kaushansky et al., Stem Cells,15(1); 97-102, 102-103, 1997; Kaushansky et al., Ann Intern Med.,126(9); 731-733, 1997; Kaushansky et al., Leukemia, 11(3); 426-427,1997; Kaushansky et al., Annu Rev Med., 48; 1-11, 1997). It wasconfirmed that recombinant TPO has ability to amelioratethrombocytopenia in animal models so that showed the potential use fortherapeutic purpose. The safety and efficacy of TPO was approved inPhase I clinical trials (Fanucchi et al., N Engl J. Med., 336(6);404-409, 1997; Basser et al., Lancet., 348(9037); 1279-1281, 1996;O'Malley et al., Blood, 88(9); 3288-3298, 1996). It also appears thatTPO could be used to enhance the recovery of platelet production inmyelosuppressed patients undergoing cancer chemotherapy.

Particularly, Escherichia coli cells transformed with the pBCTPOKIDT IIvectors were deposited with the international depositary authority asKCTC 10864BP at Korea collection for type cultures (KCTC, #52 Oun-dongYuseong-gu, Daejeon, South Korea) on 17 Oct. 2005.

Among the above two vectors, the pBCKI II vectors harboring the firstregion having a length of 6 kb are more efficient than pBCKI I vectorsin targeting to the beta-casein gene in genome. And, pBCKIDT II vectorsharboring negative selection marker, DT gene, showed 4 to 5-fold highertargeting efficiency than pBCKI II vectors. In the present invention,36.6% bEF and 41.4% bESF cells were targeted with the pBCKITPOKIDT IIvectors, and the targeting efficiency was 3.3-fold higher (41.4%/12.7%)than the that of previous targeting vectors, 12.7% in goat fetalfibroblasts (SHEN Wei et al., Chinese Journal of Biotechnology, 20(3);361-365, 2004). Therefore, we confirmed that the vectors of the presentinvention are highly efficient bovine beta-casein gene targetingvectors.

In another aspect, the present invention relates to bovine somatic cellwhich is gene-targeted with the above vectors.

A cell targeted with a vector of the present invention is derived fromeukaryotes, and can be primary, secondary, or permanent cells.Preferably, the cells are derived from cattle, sheep, goats, pigs,horses, rabbits, dogs, monkeys, and so on, but not limited to. Usefultissues capable of detaching or activating cells are, but not limitedto, livers, kidneys, spleens, bones, marrows, thymuses, hearts, muscles,lungs, brains, seminal glands, ovaries, islets, intestines, ears, skins,pancreatic tissues, prostate glands, bladders, embryos, immune systems,hematopoiesis systems, and so on. And cell types are, but not limitedto, fibroblasts, epithelial cells, nerve cells, embryonic cells, livercells, ovarian follicle cells, and so on.

A transfection method of the vector includes any method of introducing anucleic acid into the cell, and the transfection can be carried outusing an appropriate technique well known in this art. The transfectionmethods include, but not limited to, electroporation, calcium phosphateco-precipitation, retroviral infection, microinjection, DEAE-dextran,cationic liposome transfections and so on. When transfection, a linearform of vectors, which is digested with a restriction enzymes, is morepreferable than a circular form vectors, is exemplary.

In particular, a beta-casein targeting-vector inserted with a humanthrombopoietin (hTPO) was introduced into bovine embryonic fibroblasts(bEF) and bovine ear skin fibroblasts (bESF) using Lipofectamine™ 2000reagent (Invitrogen). Cationic liposome interacts efficiently withnegative charged DNA and the DNA-liposome complexes are associated withthe cell membrane, thereby leading to DNA internationalization.Insertion of the gene-targeting vector into a specific gene was detectedand confirmed by a long range PCR and a southern blot assay from thecolonies survived after the treatment with antibiotics. Using theprocedures, clones derived from bESF cells targeted with hTPO at thebeta-casein gene were obtained. The number 81 cell clone was designatedas BCTPOKIbESF81 and deposited with the Korean Collection for TypeCultures (KCTC) located in #52, Oun-dong, Yusong-ku, Taejon 305-333,Republic of Korea on Nov. 10, 2004, under the accession numberKCTC-10720BP.

In another aspect, the present invention relates to embryo which isnuclear-transferred with the said bovine somatic cell.

The term, “Nuclear transfer” is a process that genetic material of thenuclear donor cells is transferred into the enucleated oocytes. Thenuclear transfer technique makes it possible to produce geneticallyidentical animal clones because genetic material of the same donor cellis transferred into the recipient cytoplast.

In another aspect, the present invention relates to a method forproducing a bovine beta-casein gene-targeted somatic cell, whichcomprises the steps of (1) introducing the bovine beta-casein genetargeting vector into a bovine somatic cells; (2) occurring homologousevents in the transfected somatic cell; and (3) selecting the bovinebeta-casein gene-targeted somatic cell.

The bovine beta-casein gene targeting vectors of the present invention,more particularly, pBCKI I, pBCKI II, pBCKIDT I, and pBCKIDT II wereoptimized to improve targeting efficiency. Accordingly, productionefficiency of the bovine somatic cell which is bovine beta-caseingene-targeted with a gene coding a desired protein can be improved byusing the above vector.

In addition, the vector in the step (1) is introduced into cells in formof linearized or deleted form lacking plasmid vector backbone.

In another aspect, the present invention relates to a method to providea method for preparing cloned transgenic cattle, which comprises thesteps of (1) introducing the above vector into a bovine somatic cell;(2) occurring homologous recombination events in the bovine somaticcell; (3) selecting the bovine beta-casein gene-targeted somatic cellwith a desired gene by homologous recombination; (4) introducing theabove gene-targeted cell into nuclear-removed bovine oocyte to producenuclear-transferred embryo; and (5) implanting the above embryo intosurrogates to produce cloned transgenic cattle.

As referred above, the invention is proper to produce clonedgene-targeted cattle wherein the invented vectors are optimized forbovine beta-casein gene-targeting.

To remove the genetic materials of the oocytes, various methods such asphysical enucleation, chemical treatment and centrifugation withCytochalasin B treatment are employed (Tatham et al., Hum Reprod.,11(7); 1499-1503, 1996). In this invention, the physical enucleationmethod using a glass micropipette was used.

A gene-targeted somatic cell is introduced into a nuclear-removed animalembryo by using the techniques such as cell fusion method andintracytoplasmic cell injection (ICCI), etc. The cell fusion method issimple and useful for large production of embryos. The ICCI methodpermits maximum exposure of a nucleus isolated from donor cells to thecytosol in recipient cytoplasts.

The fusion of somatic cell and enucleated oocyte is accomplished bychanging charges on cell surface by electric pulse. It is convenient touse an electro-cell manipulator that the pulse length and voltage areeasily controllable.

In another aspect, the present invention relates to a method forproducing a desired protein from milk, which comprises the steps ofintroducing the vector according to the present invention into an animalsomatic cell; selecting the cell targeted with a desired gene byhomologous recombination; introducing the gene-targeted cell into anuclear-removed animal embryo; implanting the embryo into amilk-producing animal to produce a transgenic animal; and producing milkfrom the transgenic animal.

The nuclear-transferred embryos are activated and developed in vitro tothe blastocyst stage and then and then implanted to recipients.

The activation of cloned embryo induces reinitiation of cell cycle,which is temporarily quiescent, whereby the cleavage of embryo ispossible. To activate a cloned embryo, the activation of cell cyclearrest factors, such as maturation-promoting factor (MPF),mitogen-activated protein (MAP) kinase, and so on, should be suppressed,wherein for the suppression of the activation, the increase ofintracellular calcium ion in a cloned embryo is necessary. Theactivation of cell can be accomplished by the dramatic increase ofcalcium influx induced by electro-stimulation or chemical treatment suchas ionomycin, 6-dimethylaminopurine (6-DMAP), and so on, wherein theabove methods can be used independently or together. In the presentinvention, the reconstructed embryos were treated with ionomycine+6-DMAPand developed in vitro to a blastocyst stage.

As a result, cloned offspring which can express desired proteins duringlactation period are generated. Using the transgenic animals produced bythe targeting vector systems of the present invention, desired proteinscan be obtained in large quantity from the milk without causing severelethality during the embryonic or post-natal development.

In another aspect, the present invention relates to a method to obtaindesired protein from milk of the cloned transgenic cattle produced usingabove method.

The desired proteins expressed in milk can be purified usingconventional methods such as salting out (examples: ammonium sulfateprecipitation; sodium phosphate precipitation), solvent precipitation(example: protein fraction precipitation using acetone, ethanol and soon), dialysis, gel filtration, ion exchange, column chromatography suchas reverse column chromatography, ultra filtration, combinationsthereof, and so on (Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1982);Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., ColdSpring Harbor Laboratory Press(1989); Deutscher, M., Guide to ProteinPurification Methods Enzymology, vol. 182. Academic Press. Inc., SanDiego, Calif. (1990))

Hereinafter, the present invention will be described in detail referringto the following examples. However, the examples according to thepresent invention can be modified in other forms, and the scope of thepresent invention is not limited to the following examples.

EXAMPLE 1 Bovine Gene Targeting Vector Construction

1-1 pBCKI I and pBCKI II Vector Construction

In this invention, we employed a bovine beta-casein gene promoter locuswhich direct expression of foreign therapeutic genes in the milk.

The pBC 10 vector construct (FIG. 9) as previously described (Sohn etal., J. Biotechnol., 103(1); 11-19, 2003) contains a 8 kb promoter of5′-flanking sequence, the entire exon 1 and 2 kb intron 1 and 5′-UTR ofexon 2 of the bovine beta casein in a pBluescript II SK(+) plasmid(Stratagene). The beta-casein gene region of the pBC 10 vector was usedas a long arm (a first DNA region) of two invented vector cassettes,pBCKI I and pBCKI II. As shown in FIG. 10, a 10 kb beta-casein genecould be digested with Sac I and Sac II restriction enzymes and it wasused as a long arm (a first DNA sequence) of the pBCKI I vector cassette(FIGS. 10 and 14). A 6 kb beta-casein promoter region digested with AatII and Sac II restriction enzymes, including 4 kb promoter, untranslatedexon 1, 2 and intron 1 of the bovine beta-casein gene, was used as along arm (a first DNA sequence) of BCKI II (FIGS. 11 and 14).

The pBC3.1 vector (FIG. 12) was constructed to utilize as a short arm ofthe vector cassettes of the invention. The 3.2 kb DNA fragments of thebovine beta-casein gene sequence (4676 to 7898), containing exons 5, 6,7, 8 and introns 5, 6, 7 and partial introns 4 and 8, was amplified byPCR using bovine genomic DNA as the template. Sequences of the primersused for the PCR reaction are as follows:

Forward primer: SEQ ID No 1: 5′-attcagtcgagtggaacataaactttcagcc-3′Reverse primer: SEQ ID No 2: 5′-catatgtcgactgtgagattgtattttgact-3′

Bold characters of the primer sequences depict that some bases werechanged to generate Xho I and Sal I restriction enzyme sites.

The PCR product (FIG. 12) was digested with Xho I and Sal I restrictionenzymes and ligated into the Sal I site of pGEM-T (Promega). To generateenzyme sites to insert into pBC10 vector, the 3.2 kb bovine beta-caseingene fragment in pGEM-T was digested with Hinc II and Sal I restrictionenzymes. The resultant 3.1 kb fragment was ligated into Sal I site ofpBluescript II SK(+) to generate the BC3.1 vector.

The neo gene fragment utilized as a selection marker also was insertedinto pBC10 vector. To obtain the neo gene fragments including the SV40ori, early promoter, the SV40 early splicing region and polyadenylationregions, a pMAMneo vector (CLONTECH) was digested with Bam H Irestriction enzyme (FIG. 10). First, the resultant 2.7 kb DNA fragmentwas ligated into Bam H I site of the pBluescript II SK(+). Second, the 2kb neo gene fragment in the ligated vector was digested with Bgl II andBam H I restriction enzymes and then ligated into Bgl II site of thepSP73 (Promega). Finally, the 2 kb neo gene fragment in the pSP73 wasdigested with Bgl II and EcoR V restriction enzymes and then re-ligatedinto the Bgl II and EcoR V sites of the pBluescript II SK(+) harboringthe 0.7 kb pMAMneo gene fragment. These DNA digestion and ligation stepswere required for inserting the 2.7 kb neo gene fragment into the pBC10vector (FIG. 14).

As shown in FIG. 11, pBCKI I and pBCKI II vector cassettes wereaccomplished by assembling pneo2.7 and pBC3.1 gene fragments into thepBC10 vector. The neo gene in pBluescript II SK(+) was digested with BamH I and EcoR V restriction enzymes and then ligated into Bam H I andEcoR V sites of the pBC10 vector. The pBC3.1 vector was digested withEcoR V and Sal I restriction enzymes and then ligated into EcoR V andSal I sites of the pBC10 vector including the ligated pMAMneo genefragment to generate the PBCKI I vector cassette. The pBCKI I vectorcassette was digested with Aat II and Sal I restriction enzymes andligated into Aat II and Xho I sites of the pGEM-7Zf (Promega) togenerate the pBCKI II vector cassette. Therefore, the pBCKI I and pBCKIII vector cassettes were constructed.

1-2 pBCKIDT I and pBCKIDT II Vector Construction

A negative selection marker, DT gene, was inserted into the abovevectors (FIGS. 3 and 16). As using pBCKIDT I and pBCKIDT II vectorsharboring a negative selection marker, non-targeted cell clones arereduced because most cell clones which have integrated the vectors at arandom location are killed by toxicity of DT gene (FIGS. 3 and 16). TheDT gene utilized in the invention was isolated from pKO SelectDT vector(Lexicon Genetics, The Woodlands, Tex.). Using a restriction enzyme, RsrII, the Diphtheria toxin A chain (DT) gene with SV 40 Poly A and RNApolymerase II promoter was isolated. After klenow-filling the isolatedDT, gene, it was inserted into Hind II restriction sites of pBluescriptII KS (+) (Stratagene), resulting pKS DT vector. DT gene in the pKS DTvector was digested with Xho I and EcoR V and ligated into Xho I and PvuII sites of pSP73 vectors (Promega), resulting pSP73 DT vector (FIG.15). The DT gene in pSP73 DT vector was digested with Xho I and Sal Iand then, inserted into Sal I site of pBCKI I vector cassette, resultingpBCKIDT I vector. The pBCKIDT I vector was digested with Aat II and SalI and ligated into Aat II and XhoI sites of pGEM-7Zf (+) (Promega),resulting pBCKIDT II vector cassette (FIG. 16).

The fidelity of the targeting-vector cassettes was confirmed again byDNA sequencing and enzyme mapping on all regions of synthetic DNAs andall junctions of the ligated DNA fragments.

EXAMPLE 2 Construction of pBCTPOKI I and pBCTPOKI II

As shown in FIGS. 17, 18 and 19, the 1 kb Human TPO cDNA plus 300 bpbovine growth hormone was inserted pBCKI I, pBCKI II, and pBCKIDT IIvector cassettes. The 1.3 kb foreign gene harboring Sac II and Not Isites was ligated into the Sac II and Not I sites of the pBCKI, pBCKIII, and pBCKIDT II vectors, respectively, thereby generating thetargeting vectors, named as pBCTPOKI I, pBCTPOKI II, and pBCTPOKIDT II(FIGS. 17, 18, and 19). Particularly, Escherichia coli cells transformedwith the pBCTPOKIDT II vectors were deposited with the Korean Collectionfor Type Cultures (KCTC) located in #52, Oun-dong, Yusong-ku, Taejon305-333, Republic of Korea on Oct. 17, 2005, under the accession numberKCTC 10864BP.

EXAMPLE 3 Transfection of pBCTPOKI I, pBCTPOKI II, and pBCTPOKIDT IIVector Constructs into Bovine Embryonic Fibroblasts (bEF) and Bovine EarSkin Fibroblasts (bESF)

3-1 Introduction of Linearized Vectors

The plasmid DNAs, pBCTPOKI I and, pBCTPOKI II, were purified by using“QIAfilter Plasmid Midi kits” (Qiagen) and then closed circular DNAswere isolated by an equilibrium centrifugation in CsCl-ethidium bromidegradient. The isolated pBCTPOKI I and pBCTPOKI II plasmids werelinearized by digestion of Sal I (FIG. 20) and Aat II (FIG. 21)restriction enzymes, respectively, and then purified by ethanolprecipitation. Finally, the concentrations of the purified DNAs weredetermined by using a spectrophotometer.

The linearized pBCTPOKI I and pBCTPOKI II constructs were introducedinto the bEF and bESF at passage 2 or 3 by using Lipofectamine 2000reagent (Invitrogen). For bEF, we tested three different transfectionreagent volumes such as 2, 4 and 10 μl, and two different DNAconcentrations such as 2 and 4 pg. For bESF, 2, 4 and 10 μl transfectionreagent volumes and 1, 2 and 4 μg DNA concentrations were tested. Thesomatic cells were exposed to the transfection reagent-DNA complexes for24 h.

The somatic cells were cultured in Dulbecco Modified Eagle Medium(Gibco, Invitrogen corporation) supplemented with 10% FBS (Hyclone),0.001% Gentamicine (Gibco, Invitrogen corporation) and 1% MEMNon-Essential Amino Acid (Gibco, Invitrogen corporation) and the culturemedium was exchanged everyday. The cells were incubated at 37° C., 5%CO₂ in air. The volume of cell culture medium was different depending onthe plate types used as shown below.

96 well 48 well 24 well 12 well 6 well 100 mm dish 0.2 ml 0.5 ml 1.0 ml1.5 ml 3 ml 10 ml

The cells grown to confluence were treated with 1× Trypsin-EDTA (Gibco,Invitrogen corporation) solution at 37° C. for 3 min and then washedtwice with Dulbecco's Phosphate-Buffered Saline (Gibco, Invitrogencorporation) Cell colonies detached by 1× Trypsin-EDTA treatment weredissociated by gentle pipetting and then transferred into the more widerplates for proliferation. The volumes of 1× Trypsin-EDTA solution useddepends on cell culture plate types as shown below

96 well 48 well 24 well 12 well 6 well 100 mm dish 50 μl 100 μl 150 μl200 μl 500 μl 1 mlThe procedure of the experiment is shown below:

Day 1 5.5 × 10⁵ bEF cells and 3.6 × 10⁵ bESF cells at passage 2 or 3onto 6-well culture plates were transfected with the linearised BCTPOKII and BCTPOKI II DNAs using Lipofectamine 2000 reagent (Invitrogen) in 2ml of OPTIMEM I Reduced Serum Medium (Gibco, Invitrogen corporation)according to the procedure recommended by the manufacturers. Day 2Transfected cells were split into two 100 mm culture dish and furthercultured. Day 3 0.8-1.5 mg/ml G418 (Gibco, Invitrogen coporation) wasadded to the culture medium. Day Colonies of, approximately, 2-3 mm indiameter were 4-14 picked and individually replated into wells of 96well culture plates.

The cells grown until confluent in 96-well plates were transferred to 48well culture plates and then were gradually expanded in 24 well, 12well, 6 well and 100 mm culture plates.

In 6 well plates, approximately half of the transfected cells werepicked for PCR reactions and the other was transferred into two wells ofa 6 well culture plate.

3-2 Introduction of Linearized Vectors after Deleting Plasmid Vectors

The plasmid vector regions of pBCTPOKI II and pBCTPOKIDT II vectorconstructs were deleted by digesting with restriction enzymes, Aat IIand Cla I, and then, transfected into bESF and bEF cells as referredabove (FIGS. 22 and 23).

EXAMPLE 4 Identification of the Transfected Cell Clones by PCR Analysis

To screen transgenic cell lines, G418 resistant-colony cells wereanalyzed by PCR. Genomic DNAs were purified from half of the cells in 6well plates using “AccuPrep Genomic DNA Extraction Kit” (Bioneer) andPCR was carried out using “AccuPower PCR Premix” (Bioneer). Primer setsfor human TPO cDNA and thermal cycling conditions are shown below:

SEQ ID No 3 Forward primer GGA GCT GAC TGA ATT GCT CCT CGT SEQ ID No 4Reverse primer CCT GAC GCA GAG GGT GGA CCC TCC

1 cylcle of 94° C. 2 min 30 cycles of 94° C. 1 min 65° C. 30 sec 72° C.45 sec 1 cycle of 72° C. 10 min

As results of the PCR, most cell clones resistant to G418 identified astransgenic dell (FIG. 17).

EXAMPLE 5 Long-Range PCR to Detect Targeted Cell Clones

Long-range PCR was employed to determine the gene-targeted cell clonesamong transgenic cell clones. Genomic DNAs from each transgenic cellclone were purified using “AccuPrep Genomic DNA Extraction Kit”(Bioneer) or exposed by 3-4 cycles of freezing in liquid nitrogen andthawing in boiling water. Long-range PCR was performed using “AccuPowerHL PCR Premix” (Bioneer).

As shown in FIG. 5, the 5′ primer was designed to bind at the 3′ end ofneo gene within the transfected vector constructs and the 3′ primer tobind at the intron 8 of bovine beta casein gene which exists in theendogenous region, not in the vector construct. The gene targeted cellclones showed represented 4 kb PCR products on 1% agarose gel. Thesequences of a primer set and thermal cycling conditions are shownbelow:

SEQ ID No 5 Forward primer: 5′-ccacacaggcatagagtgtctgc-3′ SEQ ID No 6Reverse primer: 5′-ccacagaattgactgcgactgg- 3′

1 cylcle of 92° C. 2 min 35 cycles of 92° C. 20 sec 65° C. 45 sec 68° C.3 min 1 cycle of 68° C. 10 min

Bovine-beta casein gene targeting efficiency was compared in examples3-1 and 3-2.

In example 3-1, two cell clones were identified to be gene-targeted(FIG. 28).

As shown in examples 3-1, 41 single colonies were isolated after thetransfection of the pBCTPOKI I vector into the somatic cells. Amongthese cells, 38 colonies (93%) were identified to be transgenic, but notargeted colonies (0%) were detected. 31 single colonies were isolatedafter transfection of the pBCTPOKI II vector into the somatic cells.Among these, 29 colonies(94%) were identified as transgenic cells, and 2colonies (7%) were identified to be gene targeted with BCTPOKI II vectorat the endogenous bovine beta-casein gene locus in genome (Table 1 &FIG. 28).

TABLE 1 Transfection and targeting efficiencies of BCTPOKI I and BCTPOKIII vectors Cell types BCTPOKI I BCTPOKI II No. of bEF 23 41 2 31analyzed bESF 18 29  colonies No. of bEF 22 (96%) 38 (93%)  1 (50%) 29(94%) transgenic bESF 16 (89%) 28 (97%) colonies No. of bEF 0 (0%) 0(0%) 0 2 (7%) targeted bESF 0 (0%) 2 colonies

As shown in examples 3-2, pBCTPOKI II and pBCTPOKIDT II vectors weretransfected into bEF and bESF cells and targeted cell clones wereconfirmed using long-range PCR analysis. Here, for small volume of DNAsamples, secondary PCR analysis was performed (FIGS. 27 and 29).

Thermal cycling conditions of secondary PCR reaction are same as thoseof example 5. The targeted cell clones showed represented 3.4 kb PCRproducts on 1% agarose gel. The sequences of a primer set of secondaryPCR reaction are shown below:

Forward primer: 5′-ttcactgcattctagttgtggtttgtcca-3′ SEQ ID No 8 Reverseprimer: 5′-tctaggaccaaacatcggcttactt-3′. SEQ ID No 9

As shown in FIG. 29 A, the number 5, 30, 17, 18, 20, 21, and 26 bESFcell clones transfected with pBCTPOKIDT II vectors were identified to betargeted. B shows that the number 16 bESF cell clone transfected withpBCTPOKI II vector is targeted. C is the results of long-range PCRanalyses of wild-type bovine genomic DNA used as a negative control (−)and pneoBC3.7 vector used as a positive control (+)

pBCTPOKI II and pBCTPOKIDT II vectors were transfected into bEF and bESFcells and targeting efficiencies were compared. 18.2% (10/55) bEF cellclones transfected with pBCTPOKI II vectors and 41.4% (12/29) bEF cellclones transfected with pBCTPOKIDT II vectors were identified to begene-targeted, showing targeting efficiency by the pBCTPOKIDT II vectorsis 2.3-fold (41.4%/18.2%) higher than that by the pBCTPOKI II vectors.And, 5.7% (12/212) bESF cell clones transfected with pBCTPOKI II vectorsand 36.6% (63/172) bESF cell clones transfected with pBCTPOKIDT IIvectors were identified to be gene-targeted, showing targetingefficiency by the pBCTPOKIDT II vectors is 6.4-fold (36.6%/5.7%) higherthan that by the pBCTPOK II vectors. The average targeting efficiency ofthe pBCTPOKIDT II vectors in both cell types was 4.5-fold (37.3%/8.3%)higher than that of pBCTPOKI II vectors (Table 2), indicating thepBCKIDT II vector cassettes are highly efficient bovine beta-caseingene-targeting vectors. In addition, the targeting efficiency bypBCTPOKIDT II vectors in bEF cells was 3.3-fold higher (41.4%/12.7%)than the that by previous targeting vectors, in goat fetal fibroblasts(SHEN Wei et al., Chinese Journal of Biotechnology, 20(3); 361-365,2004), indicating the invented vectors are highly efficientgene-targeting vectors.

TABLE 2 Transfection and targeting efficiencies of pBCTPOKI II andpBCTPOKIDT II vectors No. cell N0. cell Cell clones clones typesAnalyzed targeted % % pBCTPOKI II bEF 55 10 18.2% 8.3% bESF 212 12 5.7%pBCTPOKIDT II bEF 29 12 41.4% 37.3% bESF 172 63 36.6%

EXAMPLE 6 Southern Blot Analysis to Reconfirm Targeted Cell Clones

Using vectors described example 1, bovine beta-casein gene targeting wasaccomplished, following methods referred to example 3. As results,targeting efficiency was various, depending on types of vectors andtransfected cells.

Here, both cell clones targeted with pBCTPOKI II vectors, using methodsof example 3-1, were re-analyzed by the southern blotting.

Two clones identified as gene-targeted by the PCR reactions wereanalyzed again by the southern blotting. The two cell clones weretransferred to two 100 mm culture dishes respectively. Cells wereharvested from one of the two dishes, and at least 10 micrograms DNAfrom each clone was extracted and digested with EcoR 1 for 16 hr at 37°C. EcoR I-digested DNA was separated by electrophoresis through a 0.75%agarose gel at 50V in 1×TAE buffer for 16 hr. The DNA was transferredonto nylon membranes positively charged (Boehringer Mannheim) andhybridized with the probe targeting human TPO cDNA in the vectorconstructs (see FIG. 30). The probes for southern blotting were preparedwith primers of SEQ ID NO 3 and 4 following the guidelines (Roche). ThePCR reaction was carried out using “PCR DIG labeling mix” (Roche) and“Taq DNA polymerase” (QIAGEN). Thermal cycling conditions were as below:

1 cylcle of 94° C. 3 min 30 cycles of 94° C. 45 sec 52° C. 30 sec 72° C.1 min 1 cycle of 72° C. 10 min

The gene targeting of the previously identified two clones wereconfirmed again by the southern blotting (FIG. 31). The two targetedcell clones are number 81 and number 89 cell lines.

The number 81 cell clone was designated as BCTPOKIbESF81 and depositedwith the Korean Collection for Type Cultures (KCTC) located in #52,Oun-dong, Yusong-ku, Taejon 305-333, Republic of Korea on Nov. 10, 2004,under the accession number KCTC-10720BP.

EXAMPLE 7 Preparation of Bovine Somatic Cells

Experiments were conducted according to the Animal Care and UseCommittee guidelines of the National Livestock Research Institute ofKorea. Bovine embryonic fibroblasts (bEF) were isolated from fetus atDay 45 of gestation of the Holstein cow which produce over 12,000 kgmilk a year and prepared as described previously (Koo et al., BiolReprod., 63(4); 986-992, 2000). The head of the fetus was removed usingiris scissors, and soft tissues such as liver and intestine were alsodiscarded by scooping out with two watchmarker's forceps. Bovine earskin fibroblasts (bESF) were isolated from the ear skin of a 2-years-oldHolstein cow which produces over 12,000 kg milk a year. After twicewashing with PBS (Gibco BRL), the bEF and bESF were minced with asurgical blade on a 100 mm culture dish. These procedures were performedat room temperature. The minced tissues were incubated in 10 ml of 0.05%(w/v) trypsin/0.53 mM EDTA solution in an incubator at 38.5° C. for 30min. Trypsin was inactivated by adding an equal volume of cell culturemedium supplemented with 10% FBS. The cell culture medium was composedof Dulbecco modified Eagle medium supplemented with 10% FBS, 1000 unitsof penicillin and 1000 μg/ml of streptomycin (Gibco BRL). After vigorouspipetting, the supernatant was centrifuged at 150×g for 5 min. The cellswere suspended and adjusted to a final concentration of 2×10⁶ cells/ml,and then were cultured in 10 ml culture medium at 37° C. in 5% CO₂ inair in 175-cm² tissue culture flasks (Nunc, Roskilde, Denmark) untilconfluent. The bEF and bESF cells were frozen down in cold Dulbecco'sPhosphate-Buffered Saline solution supplemented with 20% FBS and 10%Dimethyl sulphoxide and frozen at −70° C. for 16 hr. The cells werestored in liquid nitrogen until transfection experiment.

To obtain the gene-targeted cell lines, DNA-transfected cells shouldsurvive for a long period of culture in vitro without morphologicalmodification and apoptosis. In this invention, BCTPOKI I and BCTPOKI IIvectors were introduced into two types of somatic cells: bEF and bESF.It was reported that ovine post-natal fibroblasts persist through longerperiods maintaining a relatively stable chromosomal karyotype in culturethan in ovine fetal fibroblasts (Williams et al., Mol Reprod Dev.,66(2); 115-125, 2003). In this invention, the bESF sustained much longernormal morphologies in in vitro culture than bEF (Table 2).

TABLE 2 Cell survival rate on passage 4 and passage 8 No. of coloniesCell type P4 P8 Survival rate bEF 149 9 6% bESF 304 51 17%

51 (17%) of 304 bESF colonies on passage 4 were expanded by passage 8,showing normal morphologies. However, in case of bEF cells, only 6%clones were cultured by passage 8. In this experiment, we obtained twogene-targeted clones from bESF cells.

EXAMPLE 8 Freezing and Thawing of Frozen Gene-Targeted Cells

When the gene-targeted cells were grown to confluent in two 100 mmculture dishes, cells of one plate were subjected to Southern blotanalysis. And half of the other plate cells were further expanded andthe other half stored in liquid nitrogen after freezing at −70° C. for16 hr. The procedures of cell sub-culturing and storing were repeated toobtain a lot of cells for use as donor cells. The cell freezing mediumconsisted of DMEM supplemented with 20% FBS and 10% dimethyl sulphoxide(DMSO).

After thawing one vial of frozen gene-targeted cells as soon aspossible, the solution was transferred into 15 ml of tube containing 9ml of cell culture medium and centrifuged 1000 rpm for 3 min. The cellpellet was resuspended in 3 ml culture medium, plated on 6-well cultureplate containing 3 ml of culture medium, and then cultured at 37° C. in5% CO₂ in air prior to nuclear transfer.

EXAMPLE 9 Nuclear Transfer

Bovine oocytes obtained from slaughterhouse ovaries were cultured in thematuration medium at 38.57° C. in 5% CO₂ in humidified air. Thematuration medium consisted of TCM-199 (Sigma Chemical Co.) with Eaglesalts and L-glutamine supplemented with 10% (v/v) FBS (Gibco BRL, GrandIsland, N.Y.), 1 μg/ml estradiol, 1 μg/ml FSH-P (Schering-Plough AnimalHealth Corp., Kenilworth, N.J.), and 25 mM NaHCO₃. After in vitromaturation, the oocytes were transferred to 500 μl of TL-Hepessupplemented with 0.1% hyaluronidase and then cumulus of the oocyteswere removed by mechanical pipetting. The zonae pellucida of denudedoocytes were partially dissected using a fine glass needle (Tsunoda etal., J Exp Zool., 240(1); 119-125, 1986). Oocytes manipulations such asenucleation and cell injection were performed using a micromanipulatorequipped with an inverted microscope (Leitz, Ernst Leitz Wetzlar GmbH,Germany). The medium used for manipulation was TL-Hepes containing 7.5μg/ml cytochalasin B. A first polar body and partial cytoplasmpresumptively containing metaphase II chromosomes were removed togetherby using a micropipette with an inner diameter of 20 μm. Singlegene-targeted cells were individually transferred to the perivitellinespace of the recipient cytoplast. The cell-cytoplast complexes wereequilibrated in a 50 μl drop of cell fusion medium for 10-20 sec andthen transferred to a fusion chamber with two electrodes 1 mm apartoverlaid with cell fusion medium. The cell fusion medium consisted of0.3 M mannitol, 0.5 mM Hepes, 0.01% BSA, 0.1 mM CaCl₂, and 0.1 mM MgCl₂.The cell-cytoplast complexes were induced to fuse with a single pulse ofdirect current of 1.6 kV/cm for 20 μsec by an Electro Cell Manipulator2001 (BTX, San Diego, Calif.). These procedures were performed at roomtemperature. Reconstructed embryos without visible somatic cells weredetermined as fused eggs 1 h after the fusion pulse. At 4 h afterelectrofusion, the fused eggs were activated with 5 μM ionomycin for 5min, followed by treatment with 2.5 mM 6-dimethyl-aminopurine in CR1aamedium (Rosenkrans et al., Biol Reprod., 49(3); 459-462, 1993)supplemented with 10% FBS for 3.5 h at 38.5° C. in 5% CO₂ in air.

EXAMPLE 10 Culture of Reconstructed Embryos

The reconstructed embryos were cultured in CR1aa medium supplementedwith 1 mM glutamine and 1× Eagle essential amino acids solution (GibcoBRL). After culture for 3 days, cleaved embryos were further cultured ineach well of a 4-well culture plate containing 750 μl CR1aa on a mouseembryonic fibroblasts monolayer (with 10% FBS) for 4 days at 38.5° C. in5% CO₂ in air (Park et al., Anim Reprod Sci., 59(1-2); 13-22, 2000).After 7 days of culture, blastocyst formation was observed.

EXAMPLE 11 PCR Analysis of Cloned Embryos

Each embryo was transferred into 20 μl of lysis buffer, which consistedof 50 mM KCl, 1.5 mM MgCl₂, 10 mM Tris-HCl pH8.5. 0.5% Nonidet P40, 0.5%Tween and 400 μg/ml Proteinase K, and incubated at 65° C. for 30 min.Proteinase K was inactivated at 95° C. for 10 min (McCreath et al.,Nature, 405(6790); 1066-1069, 2000). For the each lysed embryo, thefirst PCR was carried out with primers of SEQ IN Nos 3 and 4 using the“AccuPower PCR Premix” (Bioneer) and then the nested PCR was performedusing 1 μl of the first PCR product. The primers of IN Nos 3 and 7 wereused and thermal cycling conditions were as follows:

Nested PCR SEQ ID No 7 Reverse primer: 5′-gagacggacctgtccagaaagctg-3′

1 cylcle of 94° C. 2 min 30 cycles of 94° C. 1 min 65° C. 30 sec 72° C.45 sec 1 cycle of 72° C. 10 min

It was analyzed by PCR at various different developmental stages whetherthe cloned embryos were transgenic or not (Table 3).

TABLE 3 PCR analysis of reconstructed embryos at various differentdevelopmental stages Developmental stages 1 2 4 8 16 blasto- cell cellcell cell cell morula cyst Total No. of 3 3 6 11 2 3 5 33 analyzedembryos No. of 3 3 6 11 2 3 5 33 transgenic embryos

As a result, 33 (100%) of 33 cloned embryos were transgenic. The resultsindicate that the present invention could produce the gene-targetedcloned animals by using the gene-targeted somatic cells.

FIG. 32 shows the PCR analysis result of nuclear-transferred embryosfrom cell clone 81 (FIG. 23)

EXAMPLE 12 Long-Range PCR Analysis of a Fetal Membrane Derived from aCloned Fetus

Reconstructed embryos at the blastocyst stages were transferred torecipients by a non-surgical method. At 36 days of gestation, a fetusand fetal membrane were flushed from the uterus of the cownon-surgically using a Foley catheter (Agtech, Manhatan, Kans.). Theextracted fetus and fetal membrane derived from the fetus were culturedin vitro as described in example 7. The long-range PCR result, showing 4kb band, confirmed that cultured cells derived from the fetal membranewere accurately targeted into a beta-casein gene in genome with thevector of the present invention (FIG. 33). The procedures of genomic DNAextraction and long-range PCR analysis are the same as described inexample 5.

INDUSTRIAL APPLICABILITY

As described herein, the transgenic cattle, which are prepared byimplanting a nuclear-transferred embryo introduced with a cell targetedwith bovine beta-casein gene targeting vector, can produce a large scaleof biomedically or biotechnically valuable proteins in their milkwithout lethality during embryonic or post-natal developmental stage byunregulated expression of the foreign proteins.

1. A bovine beta-casein gene targeting vector comprising (1) a firstregion having a length of 5 to 12 kb which is homologous to the promoterand its flanking nucleic acid sequences of bovine beta-casein gene, andcomprising exon 1, intron 1, and exon 2 of bovine beta-casein gene; (2)a region for cloning a nucleic acid coding for desired proteins; (3) aregion for coding a positive selection marker; (4) a second regionhaving a length of 2.8 to 3.5 kb which is homologous to the nucleic acidsequences of bovine beta-casein gene, and comprising exon 5, 6, 7 and 8,and intron 5, 6 and 7 of bovine beta-casein gene; wherein the nucleicacid segment corresponding to the first region is located upstream tothe nucleic acid segment corresponding to the second region in the 5′-3′arrangement of beta-casein gene.
 2. The vector according to claim 1,wherein the length of the first region is 5.5 to 10 kb.
 3. The vectoraccording to claim 1, wherein the length of the second region is 3.0 to3.2 kb.
 4. The vector according to claim 1, wherein the positiveselection marker is selected from the group consisting of neomycin(Neo), hygromycin (Hyg), histidinol dehydrogenase gene (hisD) andguanine phosphosribosyltransferase (Gpt).
 5. The vector according toclaim 1, wherein the vector further comprises a region for a negativeselection marker.
 6. The vector according to claim 5, wherein thenegative selection marker is Diphtheria toxin (DT) gene.
 7. A vectoraccording to claim 1 or 5 which is pBCKI I, pBCKI II, pBCKIDT I orpBCKIDT II, is presented in FIG. 1, FIG. 2, FIG. 16, or FIG. 3,respectively.
 8. A bovine somatic cell which is beta-caseingene-targeted with the vector according to claim 1 or
 5. 9. An embryowhich is nuclear-transferred with the bovine somatic cell according toclaim
 8. 10. A method for producing a bovine beta-casein gene-targetedsomatic cell which comprises the steps of (1) introducing the bovinebeta-casein gene-targeting vector according to claim 1 or 5 into anbovine somatic cell; (2) occurring homologous recombination events inthe bovine somatic cell; and (3) selecting the bovine beta-caseingene-targeted somatic cell with a desired gene by homologousrecombination.
 11. The method according to claim 10, wherein the vectorin the step (1) is introduced into cells in form of linearized ordeleted form lacking plasmid vector backbone.
 12. A method forgenerating transgenic cattle which comprise the steps of (1) introducingthe bovine beta-casein gene-targeting vector according to claim 1 or 5into a bovine somatic cell; (2) occurring homologous recombinationevents in the bovine somatic cell; (3) selecting the bovine beta-caseingene-targeted somatic cell with a desired gene by homologousrecombination; (4) introducing the gene-targeted cell into anuclear-removed bovine embryo to produce a nuclear-transferred embryo;and (5) implanting the embryo into a recipient.
 13. A method obtaining alarge scale of desired proteins from milk of the transgenic cattle, inaccordance with the method of claim 12.